interpretation of in vitro measurements of fructosyl transferase activity: an analysis of patterns of fructosyl transfer by fungal invertase

• Published in: The New phytologist 1991-05, Vol.118 (1), p.23-34
• Main Authors: Cairns, A.J, Ashton, J.E
• Format: Article
• Language: English
• Subjects: Acetates; Analytical, structural and metabolic biochemistry; beta-fructofuranosidase; Biological and medical sciences; Biosynthesis; catalytic activity; enzyme activity; Enzymes; Enzymes and enzyme inhibitors; Fructan; Fructans; fructosyl transferase; Fundamental and applied biological sciences. Psychology; Gels; Hydrolysis; invertase; Kinetics; Poaceae; Saccharomyces cerevisiae; Sodium; SST; sucrose; Transferases; trisaccharide; Trisaccharides; yeast; Yeasts
• ISSN: 0028-646X; 1469-8137
• DOI: 10.1111/j.1469-8137.1991.tb00562.x

A commercially available invertase preparation from yeast was analysed by native gel electrophoresis and shown to contain the large and small forms of yeast invertase but no specific fructosyl transferase. Both invertase isoforms catalysed the synthesis of the trisaccharides isokestose, neokestose and kestose from 600 mol m-3sucrose at pH 5. The hydrolytic and fructosyl transferase properties of the unfractionated invertase preparation were analysed under conditions similar to those used for the analysis of sucrose: sucrose fructosyl transferase (SST) from grasses. The sucrose concentration kinetics of the transferase gave a Kmof between 224-308 mol m-3which is similar to that reported for SST from graminaceous and other plant species. The sucrose Kmof the hydrolase was estimated at between 11-34 mol m-3. The transferase and hydrolase activities of yeast invertase could be distinguished on the basis of their responses to reaction temperature and pH, and in these respects, the data reproduced the results of grass SST studies. These observed differences in properties can be explained in terms of changes in reaction sucrose concentration and the differential kinetic properties of the hydrolase and transferase activities. The two activities could not be distinguished on the basis of their temperature-stability characteristics or their responses to pyridoxal HCl. Pyridoxal HCl was shown to cause acidification of enzyme reactions and the direct effects of this compound on the transferase and hydrolase activities were thus difficult to interpret. The results are discussed with respect to the evidence for separate SST and invertase activities in mixed enzyme systems derived from grass tissues.